Up‐regulation of P2X7 receptor–mediated inhibition of GABA uptake by nerve terminals of the human epileptic neocortex

Abstract

Summary Objective Thirty percent of patients with epilepsy are refractory to medication. The majority of these patients have mesial temporal lobe epilepsy (MTLE). This prompts for new pharmacologic targets, like ATP‐mediated signaling pathways, since the extracellular levels of the nucleotide dramatically increase during in vitro epileptic seizures. In this study, we investigated whether sodium‐dependent high‐affinity γ‐aminobutyric acid (GABA) and glutamate uptake by isolated nerve terminals of the human neocortex could be modulated by ATP acting via slow‐desensitizing P2X7 receptor (P2X7R). Methods Modulation of [3H]GABA and [14C]glutamate uptake by ATP, through activation of P2X7R, was investigated in isolated nerve terminals of the neocortex of cadaveric controls and patients with drug‐resistant epilepsy (non‐MTLE or MTLE) submitted to surgery. Tissue density and distribution of P2X7R in the human neocortex was assessed by Western blot analysis and immunofluorescence confocal microscopy. Results The P2X7R agonist, 2′(3′)‐O‐(4‐benzoylbenzoyl)ATP (BzATP, 3–100 μm) decreased [3H]GABA and [14C]glutamate uptake by nerve terminals of the neocortex of controls and patients with epilepsy. The inhibitory effect of BzATP (100 μm) was prevented by the selective P2X7R antagonist, A‐438079 (3 μm). Down‐modulation of [14C]glutamate uptake by BzATP (100 μm) was roughly similar in controls and patients with epilepsy, but the P2X7R agonist inhibited more effectively [3H]GABA uptake in the epileptic tissue. Neocortical nerve terminals of patients with epilepsy express higher amounts of the P2X7R protein than control samples. Significance High‐frequency cortical activity during epileptic seizures releases huge amounts of ATP, which by acting on low‐affinity slowly desensitizing ionotropic P2X7R, leads to down‐modulation of neuronal GABA and glutamate uptake. Increased P2X7R expression in neocortical nerve terminals of patients with epilepsy may, under high‐frequency firing, endure GABA signaling and increase GABAergic rundown, thereby unbalancing glutamatergic neuroexcitation. This study highlights the relevance of the ATP‐sensitive P2X7R as an important negative modulator of GABA and glutamate transport and prompts for novel antiepileptic therapeutic targets.

Adenosine A2A receptor and ecto-5′-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE)

“…Isolation of nerve terminals (synaptosomes) from the human hippocampus followed the methodology described in previous studies using the human neocortex and hippocampi from sheep and rats [33][34][35]. To validate this methodology, the relative enrichment in synaptophysin-positive nerve terminals compared to GFAP and PSD95 immunoreactivities identifying astrocytic glial cells and postsynaptic densities, respectively, was assessed by Western blot analysis using total lysates and synaptosomal fractions of the human hippocampus.…”

“…Control brain samples were obtained from four human cadavers submitted to forensic autopsy performed within 4-7 h postmortem; experimental procedure validation and legal constrains for using forensic brain samples were discussed in a previous study from our group [33] Clinical information about the composition of control and MTLE groups is provided in Table 1. The investigation conforms to the principles outlined in The Code of Ethics of the World Medical Association (Declaration of Helsinki).…”

“…The m ethodology for i solation nerve terminals (synaptosomes) from the human hippocampus was adapted from previous studies [33][34][35] and validated here for the first time (see below; Fig. 1 Homogenates were filtered through a nylon filter (mesh size 100 μm).…”

“…Western blot analysis was performed as previously described [33,35]. Total lysates and synaptosomes of the human hippocampus were homogenized in radio-immunoprecipitation assay buffer (Tris-HCl 25 mM (pH 7.6), NaCl 150 mM, sodium deoxycholate 1 %, Triton X-100 1 %, sodium dodecyl sulfate (SDS) 0.1 %, EDTA 5 mM, and protease inhibitors).…”

“…Immunofluorescent staining and confocal microscopy analysis was performed as previously described [33,35]. Brain samples were fixed in 4 % paraformaldehyde, cryopreserved in 30 % sucrose, and stored in a tissue freezing medium at −80°C.…”